Usefulness of combined in vivo skin comet assay and in vivo skin micronucleus test

We have already found that the in vivo skin comet assay is useful for the evaluation of primary DNA damage induced by genotoxic chemicals in epidermal skin cells. The aim of the present study was to evaluate the sensitivity and specificity of the combined in vivo skin comet assay and in vivo skin micronucleus (MN) test using the same animal to explore the usefulness of the new test method. The combined alkaline comet assay and MN test was carried out with three chemicals: 4-nitroquinoline-1-oxide (4NQO), N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and benzo[a]pyrene (B[a]P). In the first experiment, we compared DNA- and chromosome-damaging effects of 3 [72, 24 and 3 hours (h) before sacrifice] and 4 applications (72, 48, 24 and 3h before sacrifice) of 4NQO, which induces dermal irritancy. The animals were euthanized and their skin was sampled for the combination test. As a result, the 4-application method was able to detect both DNA- and chromosome-damaging potential with a lower concentration; therefore, in the second experiment, MNNG and B[a]P were topically applied four times, respectively. The animals were euthanized, and then their skins were sampled for combination tests. In the alkaline comet assay, significant differences in the percent of DNA (%DNA) in the tail were observed in epidermal skin cells treated with MNNG and B[a]P. In the MN test, an increased frequency of MN cells (%MN) cells was observed by treatment with MNNG; however, there were no significant increases. In contrast, significant differences in %MN were observed by treatment with B[a]P. From these results, we conclude that the combined in vivo skin comet assay and in vivo MN test was useful because it can detect different genotoxicity with the same sampling time and reduce the number of animals used.     

Use of the in vivo skin comet assay to evaluate the DNA-damaging potential of chemicals applied to the skin

The aim of the present study was to evaluate both sensitivity and specificity of an in vivo skin comet assay using chemically treated, hairless mouse dorsal skin as a model. N-methyl-N'-nitro-N-nitrosoguanidine (MNNG, 0.0125-0.2%), 4-nitroquinoline-1-oxide (4NQO, 0.01-0.25%), mitomycin C (MMC, 0.0125-0.05%), benzo[a]pyrene (B[a]P, 0.25-2%), and 7,12-dimethylbenz[a]anthracene (DMBA, 0.25-1%) were each applied once to the dorsal skin of hairless male mice; after 3h, epidermal skin cells were isolated, and the alkaline comet assay was performed. The assay was performed after 24h for only the B[a]P and DMBA. Furthermore, B[a]P and DMBA were evaluated by alkaline comet assay using liver cells after both 3 and 24h. The mean percent of DNA (%DNA) in tail in the 0.05-0.2% MNNG and 0.1-0.25% 4NQO treatment groups was markedly higher than in the control group at 3h post-application. Although the mean %DNA values in the tail in the B[a]P and DMBA groups were the same as the controls at 3h post-application, the 2% B[a]P and 1% DMBA groups showed significantly higher values versus controls 24h after application. No significant increases in the mean %DNA in the tail were observed in the MMC group. No clear increases in %DNA in the tail were observed in the B[a]P and DMBA groups at 3 or 24h after application in the liver. These results suggest that the in vivo skin comet assay is able to accurately identify DNA-damaging potential with a skin-specific response and is a useful method to detect the DNA-damaging potential of genotoxic chemicals on the skin.     

Coffee-mediated protective effects against directly acting genotoxins and gamma-radiation in mouse lymphoma cells

The cytokinesis-block micronucleus test was performed using L5178Y mouse lymphoma cells to ascertain whether or not standard (caffeinated) instant coffee, the commonly consumed polyphenolic beverage with antioxidant activity can protect against chromosomal damage induced by the directly acting agents N-methyl-N-nitro-N-nitrosoguanidine (MNNG), mitomycin C (MMC), methyl methanesulfonate (MMS) and gamma radiation. Our results demonstrated significant reductions in the in vitro genotoxic effects of MNNG, MMC, and MMS following co-treatment of mouse lymphoma cells with standard instant coffee. Subsequently, the comet assay was carried out to assess the effect of coffee co-treatment on the level of DNA damage induced by MMS in mouse lymphoma cells. The results demonstrated a significant reduction in MMS-induced DNA damage following co-treatment with standard instant coffee. Protective effects were observed in mouse lymphoma cells which were treated with coffee immediately after exposure to gamma radiation (1 and 2 Gy). Another experiment showed protection when the mammalian cells were irradiated (0.5 and 1 Gy) midway (at 2 h) during a 4 h coffee treatment. However, the protective effect against the lower dose (0.5 Gy) was not significant. In addition we assessed the modulatory effect of coffee on MNNG-induced apoptotic frequency by flow cytometry. The results revealed only a minor influence of coffee on the frequency of apoptotic cells induced by the test compounds, rendering an increase in sensitivity for apoptosis as a reason for the reduced genomic damage an unlikely or at least incomplete explanation.     

Contribution of apoptosis to responses in the comet assay

Apoptosis, a physiological process of selected cell deletion, leads to DNA fragmentation in typical segments of 180 base pairs. DNA strand breaks are also an effect induced by genotoxic compounds. The aim of this study was to compare these two types of damaging potentials by a known genotoxic substance and an apoptosis-inducing agent in HT-29 colon adenocarcinoma cells. The cells were incubated for 24h with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), a potent DNA damage-inducing agent, staurosporine, an inhibitor of protein kinase C and apoptosis-inducing agent, and hydrogen peroxide, a source of reactive oxygen species. Apoptosis was measured with the Annexin V affinity assay which detects the translocation of phosphatidylserine (PS) from the inner to the outer leaflet of the cytoplasmic membrane, an early event in the apoptotic process. DNA damage as an end point of genotoxicity was detected by single cell microgel electrophoresis, also called "comet assay". The results show that apoptosis does not necessarily need to correlate or coincide with DNA damage observed with genotoxic substances in the comet assay. The representative apoptosis-inducing agent (staurosporine) did not induce strand breaks in the tested concentrations (0.5 and 1.0microM); genotoxic doses of the strand break inducing agent MNNG did not induce apoptosis. Therefore, the comet assay can be used as a specific test for detecting genotoxicity, and the results are not necessarily confounded by concomittant processes leading to apoptosis.     

Genotoxicity assessment in Eisenia andrei coelomocytes: a study of the induction of DNA damage and micronuclei in earthworms exposed to B[a]P- and TCDD-spiked soils

Earthworms are useful indicators of soil quality and are widely used as model organisms in terrestrial ecotoxicology. The assessment of genotoxic effects caused by environmental pollutants is of great concern because of their relevance in carcinogenesis. In this work, the earthworm Eisenia andrei was exposed for 10 and 28 days to artificial standard soil contaminated with environmentally relevant concentrations of benzo[a]pyrene (B[a]P) (0.1, 10, 50ppm) and 2,3,7,8-tetrachloro-dibenzo-para-dioxin (TCDD) (1×10(-5), 1×10(-4), 2×10(-3)ppm). Micronucleus (MNi) induction was evaluated in earthworm coelomocytes after DNA staining with the fluorescent dye DAPI. In the same cells, the DNA damage was assessed by means of the alkaline comet assay. Induction of MNi in coelomocytes, identified according to standard criteria, was demonstrated. B[a]P exposure for 10 and 28 days induced a significant increase in MNi frequency. In TCDD-treated earthworms, a significant effect on chromosomal damage was observed at all the concentrations used; surprisingly, greater effects were induced in animals exposed to the lowest concentration (1×10(-5)ppm). The data of the comet assay revealed a significant increase in the level of DNA damage in coelomocytes of earthworms exposed for 10 and 28 days to the different concentrations of B[a]P and TCDD. The results show that the comet and MN assays were able to reveal genotoxic effects in earthworms exposed even to the lowest concentrations of both chemicals tested here. The combined application in E. andrei of the comet assay and the micronucleus test, which reflect different biological mechanisms, may be suggested to identify genotoxic effects induced in these invertebrates by environmental contaminants in terrestrial ecosystems.     

Detection of oxidative DNA damage in isolated marine bivalve hemocytes using the comet assay and formamidopyrimidine glycosylase (Fpg)

Organisms in polluted areas can be exposed to complex mixtures of chemicals; however, exposure to genotoxic contaminants can be particularly devastating. DNA damage can lead to necrosis, apoptosis, or heritable mutations, and therefore has the potential to impact populations as well as individuals. Single cell gel electrophoresis (the comet assay) is a simple and sensitive technique used to examine DNA damage in single cells. The lesion-specific DNA repair enzyme formamidopyrimidine glycoslyase (Fpg) can be used in conjunction with the comet assay to detect 8-oxoguanine and other damaged bases, which are products of oxidative damage. Fpg was used to detect oxidative DNA damage in experiments where isolated oyster (Crassostrea virginica) and clam (Mercenaria mercenaria) hemocytes were exposed to hydrogen peroxide. Standard enzyme buffers used with Fpg and the comet assay produced unacceptably high amounts of DNA damage in the marine bivalve hemocytes used in this study necessitating a modification of existing methods. A sodium chloride based reaction buffer was successfully used. Oxidative DNA damage can be detected in isolated oyster and clam hemocytes using Fpg and the comet assay when the sodium chloride reaction buffer and protocols outlined here are employed. The use of DNA repair enzymes, such as Fpg, in conjunction with the comet assay expands the usefulness and sensitivity of this assay, and provides important insights into the mechanisms of DNA damage.     

In situ evaluation of the genotoxic potential of the river Nile: II. Detection of DNA strand-breakage and apoptosis in Oreochromis niloticus niloticus (Linnaeus, 1758) and Clarias gariepinus (Burchell, 1822)

This work is part of a wider eco-toxicological study proposed to evaluate the biological impact of contaminants along the whole course of the river Nile, Egypt. Here we present data on the presence of DNA strand-breaks and apoptotic cells assessed by use of comet and diffusion assays in erythrocytes of Nile tilapia (Oreochromis niloticus niloticus) and African catfish (Clarias gariepinus). The results showed high degrees of DNA damage and increased frequencies of apoptotic nuclei in blood of fish collected from downstream compared with those sampled from upstream river Nile. Qualitative analysis revealed a shift in the frequency of DNA-damage classes towards higher damage levels correlating with the increasing pollution gradient. The degree of DNA damage measured by use of comet assay and diffusion assay exhibited seasonal variations. Both fish species showed significant increases in DNA damage during the summer. The results of our study indicated that the alkaline comet assay seems to be a useful technique for in situ genotoxic monitoring. At the same time the diffusion assay is sensitive enough to detect low frequencies of apoptotic nuclei. The results reveal species-specific differences in sensitivities, suggesting that Nile tilapia may serve as a more sensitive test species compared with the African catfish. Based on the outcome of the comet and diffusion assays, it can be concluded that the water quality of the river Nile with respect to the presence of genotoxic compounds needs to be improved, especially in its estuaries. As far as we know this is the first time that the comet and diffusion assays are used for genotoxic monitoring of the river Nile.     

Detection of oxidative DNA damage in isolated marine bivalve hemocytes using the comet assay and formamidopyrimidine glycosylase (Fpg)

Organisms in polluted areas can be exposed to complex mixtures of chemicals; however, exposure to genotoxic contaminants can be particularly devastating. DNA damage can lead to necrosis, apoptosis, or heritable mutations, and therefore has the potential to impact populations as well as individuals. Single cell gel electrophoresis (the comet assay) is a simple and sensitive technique used to examine DNA damage in single cells. The lesion-specific DNA repair enzyme formamidopyrimidine glycoslyase (Fpg) can be used in conjunction with the comet assay to detect 8-oxoguanine and other damaged bases, which are products of oxidative damage. Fpg was used to detect oxidative DNA damage in experiments where isolated oyster (Crassostrea virginica) and clam (Mercenaria mercenaria) hemocytes were exposed to hydrogen peroxide. Standard enzyme buffers used with Fpg and the comet assay produced unacceptably high amounts of DNA damage in the marine bivalve hemocytes used in this study necessitating a modification of existing methods. A sodium chloride based reaction buffer was successfully used. Oxidative DNA damage can be detected in isolated oyster and clam hemocytes using Fpg and the comet assay when the sodium chloride reaction buffer and protocols outlined here are employed. The use of DNA repair enzymes, such as Fpg, in conjunction with the comet assay expands the usefulness and sensitivity of this assay, and provides important insights into the mechanisms of DNA damage.     

The Escherichia coli K-12 SOS chromotest agar spot test for simple, rapid detection of genotoxic agents

The Escherichia coli K-12 SOS chromotest is a colorimetric (beta-galactosidase induction) system for detecting genotoxic chemicals as agents which induce filamentation in response to DNA damage. The chromotest was modified from a liquid suspension assay to a simple, convenient agar spot test, which was performed in the manner of a related colorimetric prophage induction assay (BIA). Chromotest agar dishes yielded optimal results after 16-18 h incubation, presumably because of the agar growth characteristics of tester strain PQ37. Of 44 tested chemicals, nitro aromatics, cytotoxic/antitumor agents, polycyclic hydrocarbons and aflatoxins showed good activity. Alkylating agents such as MNNG and MMS were active only at high concentrations. Compounds active in both the chromotest and BIA were active at 10-100-fold lower concentrations in the chromotest. The chromotest appeared to be less effective than the Salmonella Ames mutagenicity test in the detection of diverse classes of chemical carcinogens. The chromotest may be a useful alternative to the BIA in the study of particular classes of genotoxic compounds.     

Helicobacter pylori infection can modulate the susceptibility of gastric mucosa cells to MNNG

The pathogenesis of stomach cells can be associated with their susceptibility to exogenous dietary irritants, like nitrosamines such as dimethylnitrosamines (DMNA), and to the effects of non-dietary factors, including Helicobacter pylori infection. We used N-methyl-N’-nitro N-nitrosoguanidyne (MNNG) as a surrogate agent that induces a spectrum of DNA damage similar to DMNA. Using the alkaline comet assay, we showed that antioxidants — vitamins C and E, quercetin, and melatonin — reduced the genotoxic effect of MNNG in H. pylori-infected and non-infected human gastric mucosa cells (GMCs). To compare the sensitivity of the stomach and the blood, the experiment was also carried out in peripheral blood. We observed a higher level of DNA damage induced by MNNG in H. pylori-infected than in noninfected GMCs. We did not note any difference in the efficacy of the repair of the damage in either type of GMC. H. pylori infection may play an important role in the pathogenesis of GMCs, as it can modulate their susceptibility to dietary mutagens/carcinogens, thus contributing to gastric cancer.     

Assessment of genotoxic effects of chloropyriphos and acephate by the comet assay in mice leucocytes

Two organophosphorus (OP) pesticides (chloropyriphos and acephate) and cyclophosphamide (CP) (positive control) were tested for their ability to induce in vivo genotoxic effect in leucocytes of Swiss albino mice using the single cell gel electrophoresis assay or comet assay. The mice were administered orally with doses ranging from 0.28 to 8.96 mg/kg body weight (b. wt.) of chloropyriphos and 12.25 to 392.00 mg/kg b.wt. of acephate. The assay was performed on whole blood at 24, 48, 72 and 96 h. A significant increase in mean comet tail length indicating DNA damage was observed at 24h post-treatment (P<0.05) with both pesticides in comparison to control. The damage was dose related. The mean comet tail length revealed a clear dose dependent increase. From 48 h post-treatment, a gradual decrease in mean tail length was noted. By 96 h of post-treatment the mean comet tail length reached control levels indicating repair of the damaged DNA. From the study it can be concluded that the comet assay is a sensitive assay for the detection of genotoxicity caused by pesticides.     

Assessment of the genotoxicity in toad Bufo raddei exposed to petrochemical contaminants in Lanzhou Region, China

Single cell gel electrophoresis or comet assay, micronucleus (MN) test and global DNA methylation detection were used to assess the genotoxicity in toad Bufo raddei exposed to the petrochemical (mainly oil and phenol) polluted area in Lanzhou Region (LZR) comparing with a relatively unpolluted area in Liujiaxia Region (LJXR). The results from the present study indicated that DNA damage and MN frequency in toad from LZR were significantly higher than those from LJXR at the same sampling month, whereas the degree of global DNA methylation was lower, which implies that the petrochemical contaminants at environmental level in LZR were genotoxic to B. raddei. The degree of genotoxic damage was obviously related with the extent of pollution among the three sampling months in LZR. The significantly positive correlations between DNA damage and concentrations of oil and/or phenol existed in liver cells but erythrocytes, implying that liver is more suitable as a sentinel tissue for the assessment of genotoxic impact of low-level contamination. The results from both comet assay and global DNA methylation detection on liver cells showed that the genotoxicity varied significantly with oil and/or phenol concentrations, suggesting that these two methods are relatively sensitive and suitable for monitoring the genotoxicity of petrochemical pollutants on amphibians.     

A modified alkaline Comet assay for in vivo detection of oxidative DNA damage in Drosophila melanogaster

Modifications to the alkaline Comet assay by using lesion-specific endonucleases, such as formamidopyrimidine-DNA glycosylase (FPG) and endonuclease III (ENDOIII, also known as Nth), can detect DNA bases with oxidative damage. This modified assay can be used to assess the genotoxic/carcinogenic potential of environmental chemicals. The goal of this study was to validate the ability of this modified assay to detect oxidative stress-induced genotoxicity in Drosophila melanogaster (Oregon R(+)). In this study, we used three well known chemical oxidative stress inducers: hydrogen peroxide (H(2)O(2)), cadmium chloride (CdCl(2)) and copper sulfate (CuSO(4)). Third instar larvae of D. melanogaster were fed various concentrations of the test chemicals (50-200μM) mixed with a standard Drosophila food for 24h. Alkaline Comet assays with and without the FPG and ENDOIII enzymes were performed with midgut cells that were isolated from the control and treated larvae. Our results show a concentration-dependent increase (p<0.05-0.001) in the migration of DNA from the treated larvae. ENDOIII treatment detected more oxidative DNA damage (specifically pyrimidine damage) in the H(2)O(2) exposed larvae compared to FPG or no enzyme treatment (buffer only). In contrast, FPG treatment detected more oxidative DNA damage (specifically purine damage) in CuSO(4) exposed larvae compared to ENDOIII. Although previously reported to be a potent genotoxic agent, CdCl(2) did not induce more oxidative DNA damage than the other test chemicals. Our results show that the modified alkaline Comet assay can be used to detect oxidative stress-induced DNA damage in D. melanogaster and thus may be applicable for in vivo genotoxic assessments of environmental chemicals.     

Comet assay using mullet (Mugil sp.) and sea catfish (Netuma sp.) erythrocytes for the detection of genotoxic pollutants in aquatic environment

The development of comet assay for aquatic organisms is of particular relevance in light of the importance of coastal fisheries to several countries around the world. Two of the most common fish species native to southern Brazil are the gray mullet (Mugil sp.) and sea catfish (Netuma sp.) for which we have produced a standardized comet assay using whole erythrocytes taken from samples of these fish. We investigated the potential of the comet assay for monitoring genotoxicity in mullet and sea catfish and made a preliminary investigation of the baseline levels of DNA damage in the erythrocytes of samples of these fish from non-polluted areas as well as assessing the in vitro sensitivity of erythrocyte exposed to 2, 4 and 8 x 10(-5) M of methyl methanesulfonate (MMS) for 1, 2, 6 and 24h at 25 and 37 degrees C. Our results show that there was an increase in baseline DNA damage at higher temperatures and that the amount of MMS-induced DNA damage also increased at higher temperatures and that there was a clear dose/time response to treatment with MMS. To assess the possibility of using fish for environmental biomonitoring we also used the comet assay to investigate the in vitro genotoxic effect of MMS on whole blood cells from human donors and found a clear concentration-related effect at all exposure times, findings which agree with those of other workers. This study demonstrates the potential application of the comet assay to erythrocytes of mullets and sea catfish. However, these findings also suggest that temperature could alter both baseline DNA damage in untreated animals and in vitro cell sensitivity towards genotoxic pollutants.     

Acrylamide and glycidamide: genotoxic effects in V79-cells and human blood

Acrylamide (AA) can be formed in certain foods by heating, predominantly from the precursor asparagine. It is a carcinogen in animal experiments, but the relevance of dietary exposure for humans is still under debate. There is substantial evidence that glycidamide (GA), metabolically formed from AA by Cyp 2E1-mediated epoxidation, acts as ultimate mutagenic agent. We compared the mutagenic potential of AA and GA in V79-cells, using the hprt mutagenicity-test with N-methyl-N'-nitro-N-nitroso-guanidine (MNNG) as positive control. Whereas MNNG showed marked mutagenic effectivity already at 0.5 microM, AA was inactive up to a concentration of 10 mM. In contrast, GA showed a concentration dependent induction of mutations at concentrations of 800 microM and higher. Human blood was used as model system to investigate genotoxic potential in lymphocytes by single cell gel electrophoresis (comet assay) and by measuring the induction of micronuclei (MN) with bleomycin (BL) as positive control. AA did not induce significant genotoxicity or mutagenicity up to 6000 microM. With GA, concentration dependent DNA damage was observed in the dose range of 300-3000 microM after 4 h incubation. Significant MN-induction was not observed with AA (up to 5000 microM) and GA (up to 1000 microM), whereas BL (4 microM) induced significantly enhanced MN frequencies. Thus, in our systems GA appears to exert a rather moderate genotoxic activity.     

Comparative study of DNA damage and repair in head and neck cancer after radiation treatment

We compared DNA damage and the efficacy of its repair after genotoxic treatment with γ-radiation of lymphocytes and tissue cells isolated from patients with squamous cell carcinoma of head and neck (HNSCC) and healthy donors. Thirty-seven subjects with HNSCC and 35 healthy donors were enrolled in the study. The extent of DNA damage including oxidative lesions and efficiency of the repair were examined by alkaline comet assay. HNSCC cancer cells were more sensitive to genotoxic treatment and displayed impaired DNA repair. In particular, lesions caused by γ-radiation were repaired less effectively in metastasis of HNSCC than in healthy controls. The differences in radiation sensitivity of cancer and control cells suggested that DNA repair might be critical for HNSCC treatment. We conclude that γ-radiation might be considered as an effective therapeutic strategy for head and neck cancers, including patients in advanced stage of the disease with clear evidence of metastasis.     

Characterization ofgltA: luxCDABE fusion inEscherichia coli as a toxicity biosensor

The use ofgltA gene, as a new biomarker for environmental stress biomonitoring, was investigated because of its key position as the first enzyme of the tricarboxylic acid (TCA) cycle. A recombinant bioluminescentEscherichia coli strain, EBJM2, was constructed using a plasmid carrying the citrate synthase (gltA) promoter transcribing thePhotorhabdus luminescens luxCDABE genes (gltA::luxCDABE). The responses from this strain were studied with five different classes of toxicants: DNA damage chemicals, phenolics, oxidative-stress chemicals, PAHs, and organic solvents. EBJM2 responded strongly to DNA damage chemicals, such as mitomycin C (MMC) and methyl-nitro-nitrosoguanidine (MNNG), and nalidixic acid with the strongest responses. In contrast, tests with several compounds from the other four classes of toxicants gave no significant response. Therefore, EBJM2 was found to be sensitive to DNA damage chemicals.     

Validation of single cell gel assay in human leukocytes with 18 reference compounds

To validate the alkaline single cell gel (SCG) assay as a tool for the detection of DNA damage in human leukocytes, we investigated the in vitro activity of 18 chemicals. Thirteen of these chemicals (pyrene (PY), benzo(a)pyrene (BaP), cyclophosphamide (CP), 4-nitroquinoline-1-oxide (4NQO), bleomycin (BLM), methylmercury chloride (MMC), mitomycin C (MTC), hydrogen peroxide (HP), diepoxybutane (DEB), glutaraldehyde (GA), formaldehyde (FA), griseofulvin (GF), sodium azide (NA)) are genotoxic in at least one cell system, while five compounds (ascorbic acid (AA), glucose (GL), D-mannitol (MAN), O-vanillin (VAN), chlorophyllin (CHL)) are classified as non-genotoxic. In this in vitro SCG assay, PY, BaP and CP were positive with exogeneous metabolic activation (rat S9 mix) while 4NQO, BLM, MMC, MTC, hydrogen peroxide, and diepoxbutane were positive in the absence of metabolic activation. CHL and VAN were unexpectedly found to induce a dose-dependent increase in DNA migration. AA, GL, and MAN were negative in a non-toxic range of doses. GF gave equivocal results, while FA and GA increased DNA migration at low doses and decreased DNA migration at higher doses. This behaviour is consistent with the known DNA damaging and crosslinking properties of these compounds. These data support the sensitivity and specificity of this assay for identifying genotoxic agents.     

The comet assay differentiates efficiently and rapidly between genotoxins and cytotoxins in quiescent cells

Our main aim was to establish the efficiency of the single cell electrophoresis technique for differentiating between drugs that bind DNA and those that do not. The alkaline comet assay was used to test the responses of human leukocytes (quiescent cells) to damage induced by reportedly genotoxic and reportedly cytotoxic agents. Incubation of G0 leukocytes for 1 h with the genotoxic agents camptothecin and actinomycin C provoked DNA migration, observed as comet figures. On the other hand, when cells were treated with the cytotoxic agents cordycepin, fluorodeoxyuridine and puromycin, the leukocyte nuclei were indistinguishable from those of untreated cells. In addition, we have developed a rapid method using non-proliferating cells that requires neither culture nor lymphocyte isolation. This method promises to be useful as a rapid in vitro screening assay.